Kaposi's Sarcoma is a disfiguring and potentially fatal form of hemorrhagic sarcoma. It is characterized by multiple vascular tumors that appear on the skin as darkly colored plaques or nodules. At the histological level, it is characterized by proliferation of relatively uniform spindle-shaped cells, forming fascicles and vascular slits. There is often evidence of plasma cells, T cells and monocytes in the inflammatory infiltrate. Death may ultimately ensue due to bleeding from gastrointestinal lesions or from an associated lymphoma. (See generally Martin et al., Finesmith et al.)
Once a relatively obscure disease, it has leapt to public attention due to its association with AIDS. As many as 20% of certain AIDS-affected populations acquire Kaposi's during the course of the disease. Kaposi's Sarcoma occurs in other conditions associated with immunodeficiency, including kidney dialysis and therapeutic immunosuppression. However, the epidemiology of the disease has suggested that immunodeficiency is not the only causative factor. In particular, the high degree of association of Kaposi's with certain sexual practices suggests the involvement of an etiologic agent which is not the human immunodeficiency virus (Berel et al.).
A herpes-virus-like DNA sequence has been identified in tissue samples from Kaposi's lesions obtained from AIDS patients (Chang et al., confirmed by Ambroziuk et al.). The sequence was obtained by representational difference analysis (Lisitsyn et al.), in which DNA from affected and unaffected tissue were amplified using unrelated priming oligonucleotides, and then hybridized together to highlight differences between the cells. The sequence was partly identical to known sequences of the Epstein Barr Virus and herpesvirus saimiri. It coded for capsid and tegument proteins, two structural components sequestered in the viral interior. In a survey of tissues from various sources, the sequence was found in 95% of Kaposi's sarcoma lesions, regardless of the patients' HIV status (Moore et al. 1995a). 21% of uninvolved tissue from the same patients was positive, while 5% of samples from a control population was positive. There was approximately 0.5% sequence variation between samples.
The same sequence has been detected in body cavity lymphoma, a lymphomatous effusion with B-cell features, occurring uniquely in AIDS patients (Cesarman et al.). The copy number was higher in body cavity lymphoma, compared with Kaposi's Sarcoma. Other AIDS-associated lymphomas were negative. The sequence has also been found in peripheral blood mononuclear cells of patients with Castleman's disease (Dupin et al.). This is a condition characterized by morphologic features of angiofolicular hyperplasia, and associated with fever, adenopathy, and splenomegaly. The putative virus from which the sequence is derived has become known as Kaposi's Sarcoma associated Herpes Virus (KSHV).
Using PCR in situ hybridization, Boshoff et al. have detected KSHV polynucleotide sequences in the cell types thought to represent neoplastic cells in Kaposi's sarcoma. Serological evidence supports an important role for KSHV in the etiology of Kaposi's sarcoma (O'Leary). Kedes et al. developed an immunofluorescence serological assay that detects antibody to a latency-associated nuclear antigen in B cells latently infected with KSHV, and found that KSHV seropositivity is high in patients with Kaposi's sarcoma. Gao et al. found that of 40 patients with Kaposi's sarcoma, 32 were positive for antibodies against KSHV antigens by an immunoblot assay, as compared with only 7 of 40 homosexual men without Kaposi's sarcoma immediately before the onset of AIDS. Miller et al. prepared KSHV antigens from a body cavity lymphoma cell line containing the genomes of both KSHV and Epstein-Barr virus. Antibodies to one antigen, designated p40, were identified in 32 of 48 HIV-1 infected patients with Kaposi's sarcoma, as compared with only 7 of 54 HIV-1 infected patients without Kaposi's sarcoma.
Zhong et al. analyzed the expression of KSHV sequences in affected tissue at the messenger RNA level. Two small transcripts were found that represent the bulk of the virus specific RNA transcribed from the KSHV genome. One transcript was predicted to encode a small membrane protein; the other is an unusual poly-A RNA that accumulates in the nucleus and may have no protein encoding sequence. Messenger RNA was analyzed by cloning a plurality of overlapping KSHV genomic fragments that spanned the .about.120 kb KSHV genome from a lambda library of genomic DNA. The clones were used as probes for Northern analysis, but their sequences were not obtained or disclosed.
Moore et al. have partially characterized a KSHV genome fragment obtained from a body-cavity lymphoma. A 20.7 kb region of the genome was reportedly sequenced, although the sequence was not disclosed. 17 partial or complete open reading frames were present in this fragment, all except one having sequence and positional homology to other known gamma herpes virus genes, including the capsid maturation gene and the thymidine kinase gene. Phylogenetic analysis showed that KSHV was more closely related to equine herpes virus 2 and Saimiri virus than to Epstein Barr virus. The 20.7 kb region did not contain sequences encoding either Glycoprotein B or DNA polymerase.
The herpes virus family as a whole comprises a number of multi-enveloped viruses about 100 nm in size, and capable of infecting vertebrates. (For general reviews, see, e.g., Emery et al., Fields et al.). The double-stranded DNA genome is unusually large--from about 88 to about 229 kilobases in length. It may produce over 50 different transcripts at various stages in the life cycle of the virus. A number of glycoproteins are expressed at the viral surface, and play a role in recognition of a target cell by the virus, and penetration of the virus into the cell. These surface proteins are relatively more variant between species, compared with internal viral components (Karlin et al.). The same surface proteins are also present on defective viral particles produced by cells harboring the virus. One such non-infectious form is the L-particle, which comprises a tegument and a viral envelope, but lacks the nucleocapsid.
The herpes virus family has been divided into several subfamilies. Assignments to each of the categories were originally based on biologic properties, and are being refined as genomic sequence data emerges. The alpha subfamily comprises viruses that have a broad host range, a short replicative cycle, and an affinity for the sensory ganglia. They include the human simplex virus and the Varicella-zoster virus. The beta subfamily comprises viruses that have a restricted host range, and include Cytomegalovirus and human Herpes Virus 6. The gamma subfamily comprises viruses that are generally lymphotrophic. The DNA is marked by a segment of about 110 kilobases with a low GC content, flanked by multiple tandem repeats of high GC content. The gamma subfamily includes Epstein Barr Virus (EBV), herpes virus saimiri, equine Herpes Virus 2 and 5, and bovine Herpes Virus 4.
Herpes viruses are associated with conditions that have a complex clinical course. A feature of many herpes viruses is the ability to go into a latent state within the host for an extended period of time. Viruses of the alpha subfamily maintain latent forms in the sensory and autonomic ganglia, whereas those of the gamma subfamily maintain latent forms, for example, in cells of the lymphocyte lineage. Latency is associated with the transcription of certain viral genes, and may persist for decades until conditions are optimal for the virus to resume active replication. Such conditions may include an immunodeficiency. In addition, some herpes viruses of the gamma subfamily have the ability to genetically transform the cells they infect. For example, EBV is associated with B cell lymphomas, oral hairy leukoplakia, lymphoid interstitial pneumonitis, and nasopharyngeal carcinoma.
A number of other conditions occur in humans and other vertebrates that involve fibroproliferation and the generation of pre-neoplastic cells. Examples occurring in humans are retroperitoneal fibrosis, nodular fibromatosis, pseudosarcomatous fibromatosis, and sclerosing mesenteritis. Another condition known as Enzootic Retroperitoneal Fibromatosis (RF) has been observed in a colony of macaque monkeys at the University of Washington Regional Primate Research Center (Giddens et al.). Late stages of the disease are characterized by proliferating fibrous tissue around the mesentery and the dorsal part of the peritoneal cavity, with extension into the inguinal canal, through the diaphragm, and into the abdominal wall. Once clinically apparent, the disease is invariably fatal within 1-2 months. The condition has been associated with simian immunodeficiency (SAIDS) due to a type D simian retrovirus, SRV-2 (Tsai et al.). However, other colonies do not show the same frequency of RF amongst monkeys affected with SAIDS, and the frequency of RF at Washington has been declining in recent years.
The study of such conditions in non-human primates is important not only as a model for human conditions, but also because one primate species may act as a reservoir of viruses that affect another species. For example, the herpes virus saimiri appears to cause no disease in its natural host, the squirrel monkey (Saimiri sciureus), but it causes polyclonal T-cell lymphomas and acute leukemias in other primates, particularly owl monkeys.
There is a need to develop reagents and methods for use in the detection and treatment of herpes virus infections. The etiological linkage between KSHV and Kaposi's sarcoma, confirmed by the serological evidence, indicates the importance of this need.
For example, there is a need to develop reagents and methods which can be used in the diagnosis and assessment of Kaposi's sarcoma, and similar conditions. Being able to detect the etiologic agent in a new patient may assist in differential diagnosis; being able to assess the level of the agent in an ongoing condition may assist in clinical management. Desirable markers include those that provide a very sensitive indication of the presence of both active and latent forms viral infection, analogous to the HBsAg of Hepatitis B. Desirable markers also include those that are immunogenic, and can be used to assess immunological exposure to the viral agent as manifest in the antibody response. Glycoprotein antigens from the viral envelope are particularly suitable as markers with these characteristics. They may be expressed at high abundance near the surface not only of replicative forms of the virus, but also on L-particles produced by virally infected cells.
Second, there is a need to develop reagents and methods that can be used for treatment of viral infection--both prophylactically, and following a viral challenge. Such reagents include vaccines that confer a level of immunity against the virus. Passive vaccines, such as those comprising an anti-virus antibody, may be used to provide immediate protection or prevent cell penetration and replication of the virus in a recently exposed individual. Active vaccines, such as those comprising an immunogenic viral component, may be used to elicit an active and ongoing immune response in an individual. Antibody elicited by an active vaccine may help protect an individual against a subsequent challenge by live virus. Cytotoxic T cells elicited by an active vaccine may help eradicate a concurrent infection by eliminating host cells involved in viral replication. Suitable targets for a protective immune response, particularly antibody, are protein antigens exposed on the surface of viral particles, and those implicated in fusion of the virus with target cells.
Third, there is a need to develop reagents and methods which can be used in the development of new pharmaceuticals for Kaposi's sarcoma, and similar conditions. The current treatment for Kaposi's is radiation in combination with traditional chemotherapy, such as vincristine (Northfelt, Mitsuyasu). While lesions respond to these modalities, the response is temporary, and the downward clinical course generally resumes. Even experimental therapies, such as treatment with cytokines, are directed at the symptoms of the disease rather than the cause. Drug screening and rational drug design based upon the etiologic agent can be directed towards the long-felt need for a clinical regimen with long-term efficacy. Suitable targets for such pharmaceuticals are viral components involved in recognition and penetration of host cells. These include glycoprotein components of the viral envelope.
Fourth, there is a need to develop reagents and methods which can be used to identify new viral agents that may be associated with other fibroproliferative conditions. The representational difference analysis technique used by Chang et al. is arduously complex, and probably not appropriate as a general screening test. More desirable are a set of oligonucleotide probes, peptides, and antibodies to be used as reagents in more routine assays for surveying a variety of tissue samples suspected of containing a related etiologic agent. The reagents should be sufficiently specific to avoid identifying unrelated viruses and endogenous components of the host, and may be sufficiently cross-reactive to identify related but previously undescribed viral pathogens.